Angiogenesis is the process of developing new blood vessels that involves the proliferation, migration and tissue infiltration of capillary endothelial cells from pre-existing blood vessels. Angiogenesis is important in normal physiological processes including embryonic development, follicular growth, and wound healing as well as in pathological conditions involving tumor growth and non-neoplastic diseases involving abnormal neovascularization, including neovascular glaucoma (Folkman and Klagsbrun, Science (1987), 235:442-447).
The link between angiogenesis and cancer is well established. Neovascularization is an important step in the transition from hyperplasia to neoplasia and it must occur for tumors to grow beyond 2-3 mm in diameter and for tumor metastasis (Folkman, Nat Med (1995), 1:27-30; reviewed in Bouck et al., Adv in Cancer Res (1996), 69:135-174). A correlation between microvessel density and severity of disease has been observed in a number of different tumor types including malignant glioma (Plate & Risau, GLIA (1995), 15:339-347), and breast (Horak et al., Lancet (1992), 340:1120-124), bladder (Dickinson et al., Br J Urol (1994), 74:762-766), colon (Takahashi et al., Cancer Res (1995), 55:3964-3968), and endometrial cancer (Kirschner et al., Am J Obstet Gynecol (1996), 174:1879-1882).
Other than cancer, a number of serious diseases are associated with persistent, unregulated angiogenesis. These diseases are dominated by abnormal neovascularization. Included in the diseases in which unregulated angiogenesis is present are endometriosis, ocular disease (e.g., macular degeneration), psoiaris, and rheumatoid arthritis. Arthritis is a serious health care problem. Progressive arthritic conditions in humans cause severe pain, loss of joint mobility and disfigurement, and an overall reduction in the quality of life. In rheumatoid arthritis, the synovium hyperproliferates (aided by new blood vessels) and invades the cartilage which is destroyed.
Suppression of angiogenesis would inhibit the formation of new vessels and therefore affect tumor growth and generation of metastases. Indeed, it has been estimated that the elimination of a single endothelial cell could inhibit the growth of 100 tumor cells (Thorpe et al., Breast Cancer Research and Treatment (1995), 36:237-251). Inhibition of new capillary formation could lessen the joint destruction that occurs in rheumatoid arthritis and halt disease progression.
So far, several angiogenic factors have been identified (reviewed in Folkman, Nat Med (1995), 1:27-30; Hanahan et al., Cell (1996), 86:353-364), including the particularly potent vascular endothelial growth factor (VEGF), also known as VPF or vasculotropin (reviewed in Ferrara, Trends Cardiovasc Med (1993), 3:244-250; Ferrara and Davis-Smyth, Endocrine Rev (1997), 18:4-25). Unlike other angiogenic factors, VEGF acts as an endothelial cell-specific mitogen during angiogenesis (Terman et al., Biochem Biophys Res Commun (1992), 187:1579-1586 and Ferrara, Trends Cardiovasc Med (1993), 3:244-250). Antibodies raised against VEGF have been shown to suppress tumor growth in vivo (Kim et al., Nature (1993), 362:841-844), indicating that VEGF antagonists could have therapeutic applications as inhibitors of tumor-induced angiogenesis.
VEGF is secreted and by a number of human tumor cell lines in culture, including glioma (Tsai et al., J Neurosurg (1995), 82:864-867), melanoma (Claffey et al., Cancer Res (1996), 56:172-181), gastric cancer cells (Zhang et al., World J Gastroenterol (2002), 8 (6):994-8), Kaposi sarcoma, and epidermoid carcinoma cells (Myoken et al., Proc Natl Acad Sci USA (1991), 88:5819-5823). More importantly, VEGF transcripts or protein has been identified by in situ hybridization or immunohistochemistry in primary gliomas (Plate, et al., Lab Invest (1992), 67:529-534; Plate et al., Int J Cancer (1994), 59:520-529), hemangioblastomas (Hatva et al., Amer J Pathol (1996), 148:763-775) and breast (Toi et al., Jpn. J. Cancer Res (1994), 85:1045-1049; Anan et al., Surgery (1996), 119:333-339; Yoshiji et al., Cancer Res (1996), 56:2013-2016), colon (Brown et al., Cancer Res (1993), 53:4727-4735; Takahashi et al., Cancer Res (1995), 55:3964-3968) and renal cell tumors (Takahashi et al., Cancer Res (1994), 54:4233-4237). In glioblastoma, the message for VEGF is found in cells adjacent to necrotic regions which is consistent with upregulation by hypoxia (Shweiki et al., Nature (1992), 359, 843-845; Plate et al., Lab Invest (1992), 67:529-534). A marked increase of VEGF mRNA and protein was reported in pituitary tumors (McCabe et al., J Clin Endocrinol Metab (2002), 87 (9):4238-44) and in melanoma xenografts (Graells et al., J Invest Dermatol (2004), 123 (6):1151-61). Furthermore, patients with cancer have significantly higher serum VEGF levels than normal volunteers. The highest VEGF concentrations were observed in patients with untreated metastatic cancers.
VEGF was purified initially from the conditioned media of folliculostellate cells and from a variety of tumor cell lines (Ferrara et al., Biochem Biophys Res Commun (1989), 161:851-858; Plouet et al., EMBO J (1989), 8:3801-3806). VEGF is a homodimeric glycoprotein consisting of two 23 kD subunits and typically binds as a dimeric polypeptide to its receptors. The human gene encoding VEGF is organized into eight exons, separated by seven introns. Alternative splicing of mRNAs for the VEGF gene results in the generation of five different molecular species, having 121, 145, 165, 189, or 206 amino acid residues in the mature monomer (Tisher et al., J Biol Chem (1991), 266:11947-11954; Houck et al., Mol Endocrinol (1991), 5:1806-1814. Only VEGF165, which lacks the residues encoded by exon 6, is the mature and active form of VEGF. It binds to heparin and cell surface heparin sulfate proteoglycans, and can be expressed as a free or as a cell membrane bound form (Houck et al., 1992). VEGF206 and VEGF189 are membrane bound forms. Also, recently, a number of VEGF structural homologs have been identified: VEGF-B, VEGF-C, VEGF-D and placenta growth factor (PlGF) (Klagsbrun and D'Amore, Cytokine Growth Factor Rev (1996), 7:259-270; reviewed in Ferrara, J Mol Med (1999), 77:527-543).
Two tyrosine kinase receptors have been identified for which VEGF acts as a high affinity ligand: a fms-like tyrosine kinase-1 (Flt-1 or VEGFR-1) and a kinase domain receptor (KDR/Flk-1 or VEGFR-2) (Matthews et al., Proc Natl Acad Sci USA (1991), 88:9026-9030; Terman et al., Biochem Biophys Res Commun (1992), 187:1579-1586; De Vries et al., Science (1992), 255:989-991; Millauer et al., Cell (1993), 72:835-846). Although Flt-1 binds VEGF with 50-fold higher affinity than KDR (De Vries et al., Science (1992), 255:989-991), most of the VEGF angiogenic properties (mitogenicity, chemotaxis, and induction on morphological changes) are mediated by interaction with KDR (Waltenberger et al., J Biol Chem (1994), 269:26988-26995). Therefore, the interaction between VEGF and KDR is the most appropriate to interrupt in order to inhibit angiogenesis.
VEGF receptors typically are class III receptor-type tyrosine kinases characterized by having several, typically 5 or 7, immunoglobulin-like loops in their amino-terminal extracellular receptor ligand-binding domains (Kaipainen et al., J Exp Med (1993), 178:2077-2088). The other two regions include a transmembrane region and a carboxy-terminal intracellular catalytic domain interrupted by insertion of hydrophilic interkinase sequences of variable lengths, called the kinase insert domain (Terman et al., Oncogene (1991), 6:1677-1683).
In addition, VEGF binds to a third receptor, neuropilin-1. Neuropilin-1 (NRP-1) was first described as a co-receptor implicated in neuronal guidance that bound members of the semaphorin/collapsin family. NRP-1 is also expressed in endothelial cells and is believed to promote angiogenesis by acting as a co-receptor with VEGFR-2 (Gray et al., Cancer Res, (2005), 65 (9):3664-70). NRP-1 and VEGFR-2 do not interact directly, but are bridged by one VEGF isoform, VEGF165 (Mac Gabhann and Popel, Am J Physiol Heart Circ Physiol, (2005), 288 (6):H2851-60).
Thus, VEGF may play a broad role in a range of cancers, including cancers of the colon, rectum, renal cell (kidney), breast, non-small cell lung and ovary. Currently, Avastatin™ (bevacizumab), a therapeutic antibody developed by Genentech designed to inhibit VEGF function and thereby interfering with the blood supply to tumors has been approved as treatment for patients with metastatic cancer of the colon or rectum. Other approaches to block angiogenesis employ monoclonal antibodies specific to VEGF receptors (e.g., U.S. Pat. No. 5,955,331), compounds such as indolinone (U.S. Pat. No. 6,846,839) or peptides interacting with VEGF and thus blocking its interaction with its cognate receptor (e.g., U.S. Pat. No. 6,559,126).
However, none of the treatment options currently in clinical trials or known in the prior that block tumor-associated neovascularization by preventing VEGF binding to its cognate receptor on tumor cells, do also attempt to kill the tumor cells. This may not an easy task because, in addition to its major role in angiogenesis, VEGF affects cell survival by interfering with apoptosis (Bairey et al., Leuk Res (2004), 28 (3):243-8).
Apoptosis, or programmed cell death, is an important physiological process in multicellular organisms, both during development and for homeostasis. Apoptosis is mediated, at least in part, by a cell surface receptor protein, Fas, which plays an important role in the development and function of the immune system. Malfunction of the Fas system has been shown to cause lymphoproliferative disorders and accelerate autoimmune disorders. (Takahashi et al., Cell (1994), 76:969-976).
Fas is a type I membrane protein with a molecular weight of about 45 kD that belongs to the tumor necrosis factor (TNF) receptor family (Nagata et al., Science, 1995), 267:1449). Fas transduces apoptotic signal to the cell as a cell surface antigen. Apoptotic cell death is characterized by nuclear and cytoplasmic shrinkage, membrane blebbing, and degradation of chromosomal DNA in a characteristic pattern, and can be distinguished from necrotic cell death due to acute cellular injury.
Many tissues and cell lines weakly express Fas, but abundant expression is found in the heart, lung, liver, ovary and thymus (Watanabe-Fukunaga et al., J Immunol (1992), 148:1274). Fas transmits a signal for apoptosis or programmed cell death (Thompson, Science (1995), 267:1456) when it is triggered by binding of certain antibodies such as APO-1 (Trauth et al., Science (1989), 245:301) and anti-Fas (Yonehara et al., J Exp Med (1989), 169:1747) or the natural ligand for Fas, Fas Ligand (FasL). Fas is also expressed on the surface of tumor cells. For example, the efficiency of the induction of Fas-mediated apoptosis by anti-Fas antibodies, FasL expressing cells or recombinant FasL in tumors has been demonstrated in vivo in solid tumors implanted in mice (Timmer et al., J Pathol (2002), 196 (2):125-34).
Human, rat, and mouse FasL have been cloned (Takahashi et al., Internat Immunol (1994), 6:1567; Suda et al., Cell (1993), 75:1169; Lynch et al., Immunity (1994), 1:131; Takahashi et al., Cell (1994), 76:969). Human FasL is highly homologous to rat FasL and mouse FasL in its extracellular domain, and human FasL is capable of recognizing not only the human Fas but also the mouse Fas, and induces apoptosis. Similarly, rat and mouse FasL are capable of recognizing the human Fas and inducing apoptosis. FasL is a type II membrane protein, i.e, having an extracellular carboxyl-terminal domain and an intracellular amino-terminal domain, belongs to the TNF family of proteins and has a molecular weight of about 40 kD. (Suda et al., Cell (1993), 75:1169). The Fas ligand is strongly expressed on activated lymphocytes, in the testis (Suda et al., Cell (1993), 75:1169) and the eye (Griffith, et al., Science (1995), 270:1189), as well as on some cytotoxic T-lymphocyte (CTL) cell lines (Rouvier et al., J Exp Med (1993), 177:195).
Cells expressing FasL, as well as purified FasL protein (Suda and Nagata, J Exp Med (1994), 179:873), are cytotoxic for cells expressing Fas. Thus, FasL transmits a signal for apoptosis by binding to Fas. Also by analogy with TNF, FasL is believed to function as a trimer and presumably binds one to three Fas molecules at the interface of respective FasL units. Binding of two or more Fas molecules to a FasL trimer presumably causes oligomerization of Fas, which transmits an apoptotic signal to the Pas-expressing cell.
It would generally be desirable to be able to produce a soluble compound that combines (i) the function of a VEGFR polypeptide, i.e., binding a VEGF polypeptide, (ii) neutralizing VEGF-mediated activation of a VEGFR and thus, preventing tumor-associated neovascularization and (iii) the function of a Fas ligand in its interactions with the Fas receptor, i.e., receptor binding and/or activation of receptor mediated pathways. Such a compound would be useful for killing cancer cells that secrete VEGF and express Fas. However, a significant challenge in the recombinant protein technology has often been the expression of biologically active proteins of a transmembrane protein in the form of a soluble protein. The present invention overcomes these obstacles and meets those and other needs.